Dehydrogenases

 Pyridine nucleotide-linked dehydrogenases are enzymes that catalyze oxidation–reduction reactions involving a pyridine nucleotide and the transfer of a hydride (one proton and two electrons) from or to a second substrate molecule. Most dehydrogenase reactions are readily reversible, but some are not, and the enzymes responsible for essentially irreversible reactions are referred to as reductases; there are exceptions, however, such as formate dehydrogenase . The oxidized forms of the pyridine nucleotides are NAD (nicotinamide adenine dinucleotide) and NADP. The latter nucleotide has an additional phosphoryl group attached at the 2′-position of the ribose that is part of the adenosyl moiety (Fig. 1). The general form of a dehydrogenase reaction can be illustrated by reference to the reaction catalyzed by lactate dehydrogenase (Eq. 1): 

Not all dehydrogenase reactions are as simple as that described by Equation 1. The oxidation reactions catalyzed by isocitrate and glutamate dehydrogenases give rise to the release of CO₂ and NH₃, respectively. NAD and NADP are written frequently as NAD₊ and NADP₊ even though the overall charge on the molecule is negative in the neutral pH region; the plus refers to the charge on the nitrogen atom of the nicotinamide moiety of the oxidized forms of the pyridine nucleotides.

Dehydrogenases usually show a distinct preference for one or the other of the two pyridine nucleotides; examples are given in Table 1, and a more extensive list is available (1). The binding to the enzyme of the nonpreferred nucleotide is generally weaker, and the maximum velocity is usually lower. 

Figure 1. Structure of NADH / NADPH, showing the two prochiral hydrogen atoms at the 4-position of the nicotinamide moiety. R at the 2′-position of the ribose moiety represents either a hydrogen atom (NADH) or phosphoryl group (NADPH).

Table 1. Pyridine Nucleotide Specificity of Some Dehydrogenases

In dehydrogenase reactions (Eq. 1), the hydride is transferred to the 4-position of the nicotinamide ring of NAD or NADP (Fig. 1). The two hydrogen atoms at this position in NADH and NADPH are chemically equivalent, and the tetrahedral carbon atom is not chiral. However, the two hydrogen atoms at the 4-position are prochiral. The binding of reduced pyridine nucleotides to dehydrogenases is such that hydride transfer involves only one of the two hydrogen atoms. Thus, the enzymes exhibit regiospecificity, and hydride transfer occurs either from the pro-R hydrogen (A-side specificity) or from the pro-S hydrogen (B-side specificity). Examples of dehydrogenases that fall into each of these two classes are given in Table 1. Attempts have been made to explain why some dehydrogenases are A-side and others are B-side, but no convincing explanation has been forthcoming (2).

 The side-specificity of dehydrogenases allows the preparation of reduced pyridine nucleotides with a deuterium (D) or tritium (T) atom replacing a hydrogen atom in either the pro-R or pro-S positions. Extensive use has been made of deutero- and tritio-pyridine nucleotides to gain information about enzymic catalysis (3). The cleavage of a bond can be expected to be two to eight times slower than with a bond. With chemical reactions, the full isotope effect difference in rates is usually observed, because the bond-breaking step is rate-limiting. But with enzyme-catalyzed reactions, steps other than the catalytic one can be at least partly rate-limiting. This results in the observed isotope effect, with NADD or NADPD being smaller than the absolute value for the bond-breaking step. Indications as to where the rate-limiting steps lie for a particular dehydrogenase reaction come from the use of initial velocity studies to determine the apparent first-order rate constants k and the maximum velocities V with the protio- and deutero-substrates. The observed isotope effects are then calculated from the ratio of the values for (V/K)_H and (V/K)_D, expressed as ^D  )V/K) and the ratio of the values for V_H and V_D, expressed as ^DV. The values for these ratios usually range from 1.0 to 3.5. Low values for ^D(V/K) can result from the stickiness of substrates. Values for ^DV will be low whenever product release is rate-limiting (3). The tritiated forms, NADT and NADPT, can be used for determinations of ^T(V/K), but not from initial velocity studies, as the tritium is only a trace label. For this same reason, it is not possible to obtain values for TV. The value for ^T )V/K) will be higher than that for D(V/K) as the bond is stiffer than the bond. As ^Tk = (^Dk)1.442, the general function [^Dk – 1]/[^Dk1.442 – 1] can be used to determine the intrinsic isotope effect for a dehydrogenase reaction (4). It is this value that gives insights into the chemical mechanism of the reaction and the nature of the transition state.

 The pyridine nucleotides that function as effective substrates for most dehydrogenases have a nicotinamide–ribose linkage (Fig. 1) with a b-anomeric configuration and are referred to, for example, as b-NAD. These pyridine nucleotides also occur with an a-anomeric configuration and are found in tissue extracts, as well as commercial preparations. Both a-NADH and b-NADH undergo thermal epimerization reactions to produce an equilibrium mixture with 10% of the a form (5).  There are no examples of a dehydrogenase that specifically uses a-NADH or a-NADPH, although alcohol dehydrogenase and lactate dehydrogenases utilize a-NADH with maximum velocities some three to four orders of magnitude lower than with b-NADH. The same side-specificity is observed with both anomers of NADH (5). By contrast, a-NADPH is a relatively good substrate for the dihydrofolate reductase that is encoded by an R-plasmid (6). The K_m value of 17 µM is comparable to the value of 4 µM for b-NADPH. The maximum velocity of the reaction with a-NADPH is 70% of that with b-NADPH. It has been proposed that those dehydrogenases which show activity with both the a- and b-anomers must have sufficient conformational freedom to permit specific orientation of the dihydronicotinamide ring for the reduction of other substrates.

References

1. G. Popjak (1970) The Enzymes 2, 115–215. 

2. N. J. Oppenheimer (1984) J. Am. Chem. Soc. 106, 3032–3033. 

3. W. W. Cleland (1982) CRC Crit. Rev. Biochem. 13, 385–428. 

4.  D. B. Northrop (1975) Biochemistry 14, 2644–2651. 

5. N. J. Oppenheimer and N. O. Kaplan (1975) Arch. Biochem. Biophys. 166, 526–535.

6. S. L. Smith and J. J. Burchall (1983) Proc. Natl. Acad. Sci. 80, 4619–4623.